The present invention relates to polypeptides that regulate production of Angiotensin 1-9 via such mechanisms as, for example, inhibition of ACE-2. Such polypeptides have uses for example, in the detection, isolation, and/or purification of ACE-2 and/or Angiotensin 1-9. The invention also relates to nucleic acid molecules encoding these ACE-2 binding polypeptides, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention also relates to methods and compositions for detecting, diagnosing, or prognosing a disease or disorder associated with aberrant ACE-2 or Angiotensin 1-9 expression or inappropriate function of ACE-2.or Angiotensin 1-9, comprising ACE-2 binding polypeptides or fragments or variants thereof, that specifically regulate ACE-2 action. The present invention further relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with aberrant ACE-2 or Angiotensin 1-9 expression or inappropriate ACE-2 function or Angiotensin 1-9 function, comprising administering to an animal, preferably a human, an effective amount of one or more ACE-2 binding polypeptides or fragments or variants thereof, that specifically regulate ACE-2.
The renin-angiotensin system (RAS) plays an important role in circulatory homeostasis at both systemic and local levels. Angiotensin converting enzyme (ACE), a 175 kD protein known to be widely distributed throughout the cardiovascular system, has been long recognized as the key enzyme in the generation of angiotensin II, a peptide that regulates fluid balance, blood pressure and local blood flow in a number of tissues (Peach, M. J. Physiological Reviews 57:313-370 (1997)). As part of an ongoing strategy to establish genes associated with cardiovascular function via high throughput cDNA sequencing, we identified a member of the RAS family of enzymes, ACE-2, from human kidney. This enzyme was also identified in a variety of tissues by others (Donoghue et al., Circulation Research 87:e1-e9 (2000), Tipinis et al., The Journal of Biological Chemistry 275:33238-33243 (2000)). The unmodified ACE-2 protein contains transmembrane and signal peptide domains, but unlike ACE, ACE-2 contains just one single extracellular Zn+2 binding metalloprotease domain (Tipinis et al., The Journal of Biological Chemistry 275:33238-33243 (2000)). ACE-2 mRNA has a more limited expression pattern than ACE (Donoghue et al., Circulation Research 87:e1-e9 (2000)) and, remarkably, no detectable expression in lungs (unpublished data).
ACE-2 and related carboxypeptidases (Snyder et al., The Journal of Biological Chemistry 260:7857-7860 (1985); Kokkonen et al., Circulation 95:1455-1463 (1997)) catalyze the removal of the C-terminal leucine from angiotensin I to form the nonapeptide angiotensin 1-9 (A1-9) (SEQ ID NO:145) or des-Leu10-angiotensin I (Donoghue et al., Circulation Research 87:e1-e9 (2000); Tipinis et al., The Journal of Biological Chemistry 260:7857-7860 (2000); Snyder et al., The Journal of Biological Chemistry 260: 7857-7860 (1985); Snyder et al., Biochemica et Biophysica Acta 871:1-5 (1986)). Circulating A1-9 has been detected in vivo at levels twice that of angiotensin II (Oparil et al., Circulation Research 29 682-690 (1971); Johnson et al., Peptides 10:489-492) (1989)). In the case of ACE-2, the above reaction is not blocked by captopril, lisinopril or enalaprilat (Donoghue et al., Circulation Research 87:e1-e9 (2000); Tipinis et al., The Journal of Biological Chemistry 275:33238-33243 (2000)). The unique expression profile of ACE-2, spectum of its enzymatic activity and inhibitory effects of its product A1-9 on ACE have led to the speculation that ACE-2 functions to affect circulatory homeostasis by promoting vasodilation (Donoghue et al., Circulation Research 87:e1-e9 (2000); Snyder et al., The Journal of Biological Chemistry 260:7857-7860 (1985)). However, A1-9 has been shown to be a weak vasoconstrictor in isolated rat aorta and have weak pressor activity in anesthetized rats and dogs (Oparil et al., Circulation Research 29:682-690 (1971)). Therefore, we hypothesized that one of the physiologic roles of ACE-2 is to increase arterial pressure through the actions of its catabolic product, A1-9. As such, ACE-2 might be a valid target for drug development in hypertension.
Accordingly, molecules that specifically bind ACE-2 would find a variety of uses in the study of ACE-2, angiotensin 1-9) (SEQ ID NO:145), and angiotensin, as well as ACE, and its known substrates: Angiotensin II (SEQ ID NO:144), Angiotensin 1-7, des-Asp, bradykinin, neurotensin, and Substance P. Further, molecules that specifically bind ACE-2 would also find a variety of uses in the manufacture and purification of ACE-2, ACE, angiotensin, angiotensin II, and/or Angiotensin 1-9 in commercial and medically pure quantities, and in the development new therapeutic or diagnostic reagents. ACE-2 binding polypeptides may also find medical utility in, for example, the treatment of cardiovascular disorders (e.g., hypertension, chronic heart failure, left ventricular failure, stroke, cerebral vasospasm after subarachnoid injury, atherosclerotic heart disease, and retinal hemorrhage), renal disorders (e.g., renal vein thrombosis, kidney infarction, renal artery embolism, renal artery stenosis, myocardial hypertrophy, hypertrophy and/or hyperplasia of conduit and/or resistance vessels, myocyte hypertrophy, and fibroblast proliferative diseases), inflammatory diseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis, polytrauma, inflammatory bowl disease, acute and chronic pain, rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g., asthma, adult respiratory distress syndrome, wound healing, and scar formation), as well as several other disorders and/or diseases (e.g., periodontal disease, dysmenorrhea, premature labor, brain edema following focal injury, diffuse axonal injury, and reperfusion injury).
The present invention provides new polypeptides and families of polypeptides that specifically bind ACE-2 and/or ACE-2-like polypeptides. In particular, the invention encompasses polypeptides that specifically bind to a polypeptide or polypeptide fragment of human ACE-2 (SEQ ID NOs:138 and/or 142).
In particular, the invention relates to ACE-2 binding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-136, preferably SEQ ID NOs: 11-39, more preferably SEQ ID NOs:23-24 and 36-39, as referred to below, in Tables 1-2 and Example 1 below, and fragments and variants thereof.
In specific preferred embodiments, the ACE-2 binding polypeptides of the invention bind ACE-2 and/or ACE-2-like polypeptides with high affinity. In other embodiments, the ACE-2 binding polypeptides of the invention reversibly bind ACE-2 and/or ACE-2-like polypeptides. In still other embodiments, the ACE-2 binding polypeptides of the invention irreversibly bind ACE-2 and/or ACE-2-like polypeptides.
The cysteine residues in certain polypeptides according to the invention are believed to form a disulfide bond, which would cause the polypeptide containing these cysteine residues to form a stable loop structure under non-reducing conditions. Especially preferred ACE-2 binding polypeptides of the invention are polypeptide molecules that comprise amino acid sequences that form stable loop structures or other stable structures that bind ACE-2 or ACE-2-like polypeptides.
Preferred binding polypeptides specific for ACE-2 include two separated, invariant cyteine residues and are thus capable of forming a cyclic strucure under non-reducing conditions via a disulfide bond formed between the cysteine side chains. Specific ACE-2 binding polypeptides according to the present invention include polypeptides comprising amino acid sequences of the following general formulae I-X:
Z1-X1-A-X2-X3-C-X4-X5-F-Z2 (SEQ ID NO:1)xe2x80x83xe2x80x83I.
wherein,
Z1 is a polypeptide of at least 2 amino acids;
X1 is any amino acid except cysteine;
X2 is L or M (preferably L);
X3 is F or Y (preferably F);
X4 is F, L, M, or V (preferably V);
X5 is D or E;
Z2 is a polypeptide of at least one amino acid or is absent; and
Z1 contains at least one cysteine residue such that formation of a disulfide bond with the invariant cysteine residue (C) forms a cyclic peptide of six or ten amino acids.
Z1-X1-C-X2-X3-X4-X5 (SEQ ID NO:2)xe2x80x83xe2x80x83II.
wherein,
Z1 is a polypeptide of at least six amino acids;
X1 is F, M, W, or Y (preferably F or Y);
X2 is F, I, L, M, or V (preferably F or L);
X3 is D, E, or T (preferably D);
X4 is F or M (preferably F);
Z2 is a polypeptide of at least one amino acid or is absent; and
Z1 contains at least one cysteine residue such that formation of a disulfide bond with the invariant cysteine residue (C) forms a cyclic peptide of eight or ten amino acids.
Z1-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-C-Z2 (SEQ ID NO:3)xe2x80x83xe2x80x83III.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is A, D, F, G, H, L, N, P, or S (preferably D);
X2 is A, D, F, G, H, N, S, W, or Y (preferably D);
X3 is D, E, H, L, M, or V (preferably D or E);
X4 is D, E, G, N, R, Q, S, or V (preferably D or E);
X5 is N, T, or W (preferably W);
X6 is any amino acid except cysteine;
X7 is any amino acid except cysteine;
X8 is F, W, or Y (preferably F);
X9 is any amino acid except cysteine;
X10 is any amino acid except cysteine;
X11 is any amino acid except cysteine;
X12 is any amino acid except cysteine;
X13 is any amino acid except cysteine; and
Z2 is a polypeptide of at least one amino acid or is absent.
Z1-R-X1-X2-X3-X4-D-S-X5-C-Z2 (SEQ ID NO:4)xe2x80x83xe2x80x83III.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is any amino acid except cysteine;
X2 is any amino acid except cysteine;
X3 is C, E, or S;
X4 is K, L, or R (preferably R);
X5 is A, R, or S (preferably R);
Z2 is a polypeptide of at least one amino acid or is absent; and
wherein, if X3 is cysteine (C), then Z1 contains a C-terminal cysteine residue.
Z1-C-X1-X2-X3-D-C-X4-Z2 (SEQ ID NO:5)xe2x80x83xe2x80x83V.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is any amino acid except cysteine (preferably L, H, or M);
X2 is N or T (preferably T);
X3 is any amino acid except cystein (preferably D, M, N, or S);
X4 is V or I (preferably V);
Z2 is a polypeptide of at least one amino acid or is absent.
Z1-C-F-X1-W-X2-Z2 (SEQ ID NO:6);xe2x80x83xe2x80x83VI.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is D or E;
X2 is D or E;
Z2 is a polypeptide of at least two amino acids and contains at least one cysteine residue such that formation of a disulfide bond with the invariant cysteine residue (C) forms a cyclic peptide of seven, eight or twelve amino acids.
Z1-X1-E-X2-C-H-X3-X4-P-X5-X6-C-Z2 (SEQ ID NO:7)xe2x80x83xe2x80x83VII.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is W or Y;
X2 is any amino acid except cysteine;
X3 is W or Y;
X4 is any amino acid except cysteine;
X5 is any amino acid except cysteine;
X6 is any amino acid except cysteine; and
Z2 is a polypeptide of at least one amino acid or is absent.
Z1-K-E-C-K-F-G-Y-X1-X2-C-L-X3-W-Z2 (SEQ ID NO: 8)xe2x80x83xe2x80x83VIII.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is any amino acid except cysteine;
X2 is any amino acid except cysteine;
X3 is any amino acid except cysteine; and
Z2 is a polypeptide of at least one amino acid or is absent.
Z1-X1-X2-C-X3-X4-W-X5-X6-P-C-Z2 (SEQ ID NO:9)xe2x80x83xe2x80x83IX.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is D or H (preferably D);
X2 is any amino acid except cysteine (preferably H, N, or W);
X3 is G or is absent;
X4 is T or N (preferably T);
X5 is any amino acid except cysteine (preferably A, N, W, or Y);
X6 is any amino acid except cysteine (preferably H, N, or Q); and
Z2 is a polypeptide of at least one amino acid or is absent.
xe2x80x83Z1-C-X1-X2-X3-R-X4-X5-P-W-X6-X7-C-Z2 (SEQ ID NO:10)xe2x80x83xe2x80x83X.
wherein,
Z1 is a polypeptide of at least one amino acid or is absent;
X1 is any amino acid except cysteine (preferably K, L, R, or S);
X2 is A or P (preferably P);
X3 is any amino acid except cysteine (preferably I, L, Q, or V);
X4 is any amino acid except cysteine (preferably D, G, H, M, Q, or Y);
X5 is any amino acid except cysteine (preferably D, F, K, S, or Y);
X6 is any amino acid except cysteine (preferably F, K, M, or W; most preferably W);
X7 is any amino acid except cysteine (preferably A, F, K, R, or V); and
Z2 is a polypeptide of at least one amino acid or is absent.
ACE-2 binding polypeptides of the present invention include polypeptides comprising amino acid sequences selected from the group consisting of:
ACE-2 binding polypeptide molecules of the invention may also have an amino terminal (N-terminal) capping or functional group, such as an acetyl group, which, for example, blocks the amino terminal amino group from undesirable reactions or is useful in linking the ACE-2 binding polypeptide to another molecule, matrix, resin, or solid support. ACE-2 binding polypeptides of the invention may also have a carboxy terminal (C-terminal) capping or functional group, such as an amide group, which, for example, blocks the C-terminal carboxyl group from undesirable reactions or provides a functional group useful in conjugating the binding polypeptide to other molecules, matrices, resins, or solid supports. Preferably, the N- and/or C-terminal capping groups are polypeptide linker molecules. An especially preferred C-terminal linker molecule that is useful for immobilizing an ACE-2 binding polypeptide of the invention to a solid support or chromatographic matrix material comprises the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:146).
The invention also encompasses ACE-2 binding polypeptides that have been modified, for example, to increase or decrease the stability of the molecule, while retaining the ability to bind ACE-2 and/or ACE-2-like polypeptides. An example of a modified ACE-2 binding polypeptide of the invention is a polypeptide in which one of two cysteine residues is substituted with a non-naturally occurring amino acid that is capable of condensing with the remaining cysteine side chain to form a stable thioether bridge, thereby generating a cyclic BLyS binding polypeptide. Such cyclic thioether molecules of synthetic peptides may be routinely generated using techniques known in the art, e.g., as described in PCT publication WO 97/46251, incorporated herein by reference.
In another embodiment, the invention provides ACE-2 binding polypeptides of the invention attached, coupled, linked or adhered to a matrix or resin or solid support. Techniques for attaching, linking or adhering polypeptides to matrices, resins and solid supports are well known in the art. Suitable matrices, resins or solid supports for these materials may be any composition known in the art to which an ACE-2 binding polypeptide of the invention could be attached, coupled, linked, or adhered, including but not limited to, a chromatographic resin or matrix, such as SEPHAROSE-4 FF agarose beads, the wall or floor of a well in a plastic microtiter dish, such as used in an enzyme-liked immunosorbent assay (ELISA), or a silica based biochip. Materials useful as solid supports on which to immobilize binding polypeptides of the invention include, but are not limited to, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. An ACE-2 binding polypeptide of the invention may be immobilized on a matrix, resin or solid support material by a non-covalent association or by covalent bonding, using techniques known in the art. Preferably, an ACE-2 binding polypeptide of the invention is immobilized on a chromatography material such as SEPHAROSE-4 FF agarose. In an even more preferred embodiment, an ACE-2 binding polypeptide of the invention is coupled to a chromatography material using a linker molecule. A preferred linker molecule according to the present invention is a polypeptide comprising the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:146). Most preferably, the affinity chromatography material of the invention comprises an ACE-2 binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:11-136, which is linked to a chromatography material by a polypeptide linker molecule having the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:146). ACE-2 binding polypeptides of the invention attached, coupled, linked or adhered to a matrix or resin or other solid support are useful for methods of detecting, isolating and purifying ACE-2 and/or ACE-2-like polypeptides as well as Angiotensin 1-9 and/or Angeiotensin 1-9-like polypeptides, particularly for purification of ACE-2 and/or ACE-2-like polypeptides as well as Angiotensin 1-9 and/or Angeiotensin 1-9-like polypeptides by affinity chromatography.
In certain preferred embodiments, the ACE-2 binding polypeptides of the present invention or phage displaying such binding polypeptides, irreversibly bind the ACE-2 protein in its native form.
In certain preferred embodiments, the ACE-2 binding polypeptides of the present invention or phage displaying such binding polypeptides, reversibly bind the ACE-2 protein in its native form.
In a further embodiment, the present invention encompasses a composition of matter comprising isolated nucleic acids, preferably DNA, encoding an ACE-2 binding polypeptide of the invention. In a specific embodiment, nucleic acid molecules of the invention encode an ACE-2 binding polypeptide of the invention as provided in SEQ ID NOs: 1-136. In additional embodiments, nucleic acid molecules of the invention encode a polypeptide variant or fragment of a polypeptide comprising an amino acid sequence of SEQ ID NOs: 1-136. In a further additional embodiment, nucleic acid molecules of the invention encode an ACE-2 binding polypeptide, the complementary strand of which nucleic acid hybridizes to a polynucleotide sequence encoding a polypeptide described in Tables 1-2 and in Example 1 (SEQ ID NOs: 1-136), under stringent conditions, e.g., hybridization to filter-bound DNA in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C. followed by one or more washes in 0.2xc3x97SSC/0.1% SDS at about 50-65xc2x0 C., under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6xc3x97SSC at about 45xc2x0 C. followed by one or more washes in 0.1xc3x97SSC/0.2% SDS at about 68xc2x0 C., or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds. , 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules encoding the ACE-2 binding polypeptides of the present invention (as well as fragments and variants thereof), and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells. The invention further provides for the use of such recombinant vectors in the production of ACE-2 binding polypeptides by recombinant techniques.
The ACE-2 binding polypeptides, nucleic acids, transformed host cells, and genetically engineered viruses and phage of the invention (e.g., recombinant phage), have uses that include, but are not limited to, the detection, isolation, and purification of ACE-2.
In another embodiment of the invention, recombinant bacteriophage displaying ACE-2 binding polypeptides on their surfaces are also provided. Such phage may be routinely generated using techniques known in the art and are useful, for example, as screening reagents and reagents for detecting ACE-2.
In another embodiment, an ACE-2 binding polypeptide of the invention is used to detect or isolate ACE-2 or ACE-2-like polypeptides in a solution. Such solutions include, but are not limited to, ACE-2 or ACE-2-like polypeptides suspended or dissolved in water or a buffer solution as well as any fluid and/or cell obtained from an individual, biological fluid, body tissue, body cell, cell line, tissue culture, or other source which may contain ACE-2 or ACE-2-like polypeptides, such as, cell culture medium, cell extracts, and tissue homogenates. Biological fluids include, but are not limited to, sera, plasma, lymph, blood, blood fractions, urine, synovial fluid, spinal fluid, saliva, and mucous.
In another embodiment, the present invention provides a method for detecting ACE-2 protein and/or ACE-2-like polypeptide in a solution comprising, contacting the solution with an ACE-2 binding polypeptide of the invention and detecting binding of ACE-2 or ACE-2-like polypeptide to the ACE-2 binding polypeptide. The ACE-2 binding polypeptide may be either free or immobilized. Preferably, the ACE-2 binding polypeptide is a polypeptide immobilized on a solid surface or chromatographic material or the well of a plastic microtiter assay dish.
Another embodiment of the present invention is a method for isolating ACE-2 protein and/or an ACE-2-like polypeptide from a solution, comprising:
(a) contacting the solution with an ACE-2 binding polypeptide under conditions that permit binding of the ACE-2 and/or ACE-2-like polypeptides to ACE-2 binding polypeptides, and
(b) recovering the ACE-2 and/or ACE-2-like polypeptides.
A further embodiment of the present invention is a method for isolating ACE-2 protein and/or an ACE-2-like polypeptide from a solution, comprising:
(a) contacting the solution with an ACE-2 binding polypeptide under conditions that permit binding of the ACE-2 and/or ACE-2-like polypeptides to ACE-2 binding polypeptides,
(b) separating the complex(es) formed by the ACE-2 binding polypeptide and ACE-2 and/or ACE-2-like polypeptides from other components of the solution,
(c) dissociating the ACE-2 binding polypeptide from the ACE-2 and/or ACE-2 -like polypeptides, and
(d) recovering the dissociated ACE-2 and/or ACE-2-like polypeptides.
In another embodiment, the invention provides kits containing a binding polypeptide of the invention for use in methods of detecting or isolating ACE-2 and/or ACE-2-like polypeptides.
The present invention also provides panels of ACE-2 binding polypeptides (including molecules comprising, or alternatively consisting of, ACE-2 binding polypeptide fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different ACE-2 binding polypeptides of the invention. The present invention further provides mixtures of ACE-2 binding polypeptides, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different ACE-2 binding polypeptides of the invention. The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more ACE-2 binding polypeptides of the present invention (including molecules comprising, or alternatively consisting of, ACE-2 binding polypeptide fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more ACE-2 binding polypeptides or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more ACE-2 binding polypeptides of the invention.
The present invention further provides for fusion proteins comprising an ACE-2 binding polypeptide (including molecules comprising, or alternatively consisting of, ACE-2 binding polypeptide fragments or variants thereof) of the invention, and a heterologous polypeptide. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.
The present invention also encompasses methods and compositions for detecting, diagnosing, prognosing, and/or monitoring diseases or disorders associated with aberrant ACE-2 or ACE expression or inappropriate ACE-2 or ACE receptor function in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, use of ACE-2 binding polypeptides (including molecules which comprise, or alternatively consist of, ACE-2 binding polypeptide fragments or variants thereof) that specifically bind ACE-2. Diseases and disorders which can be detected, diagnosed, prognosed and/or monitored with the ACE-2 binding polypeptides of the invention include, but are not limited to, cardiovascular disorders (e.g., hypertension, chronic heart failure, left ventricular failure, stroke, cerebral vasospasm after subarachnoid injury, atherosclerotic heart disease, and retinal hemorrhage), renal disorders (e.g., renal vein thrombosis, kidney infarction, renal artery embolism, renal artery stenosis, and edema, hydronephritis), proliferative diseases or disorders (e.g., vascular stenosis, myocardial hypertrophy, hypertrophy and/or hyperplasia of conduit and/or resistance vessels, myocyte hypertrophy, and fibroblast proliferative diseases), inflammatory diseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis, polytrauma, inflammatory bowl disease, acute and chronic pain, rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g., asthma, adult respiratory distress syndrome, wound healing, and scar formation), as well as several other disoders and/or diseases (e.g., periodontal disease, dysmenorrhea, premature labor, brain edema following focal injury, diffuse axonal injury, and reperfusion injury).
In specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing, prognosing and/or monitoring diseases or disorders for preventing, treating and/or ameliorating diseases or disorders associated with hypertension (e.g., accelerated hypertension, renal failure, vascular accidents, myocaridal infarction, and stroke).
In other specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing, prognosing and/or monitoring diseases or disorders associated with hypotension (e.g., shock, intracranial hypotension, and syncope).
The present invention further encompasses methods and compositions for preventing, treating and/or ameliorating diseases or disorders associated with aberrant ACE-2 or ACE expression or inappropriate ACE-2 or ACE function in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, administering to an animal in which such treatment, prevention or amelioration is desired one or more ACE-2 binding polypeptides (including molecules which comprise, or alternatively consist of, ACE-2 binding polypeptide fragments or variants thereof) in an amount effective to treat, prevent or ameliorate the disease or disorder. Diseases and disorders which can be prevented, treated, and/or ameliorated with the ACE-2 binding polypeptides of the invention include, but are not limited to, cardiovascular disorders (e.g., hypertension, chronic heart failure, left ventricular failure, stroke, cerebral vasospasm after subarachnoid injury, atherosclerotic heart disease, and retinal hemorrhage), renal disorders (e.g., renal vein thrombosis, kidney infarction, renal artery embolism, renal artery stenosis, and edema, hydronephritis), proliferative diseases or disorders (e.g., vascular stenosis, myocardial hypertrophy, hypertrophy and/or hyperplasia of conduit and/or resistance vessels, myocyte hypertrophy, and fibroblast proliferative diseases), inflammatory diseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis, polytrauma, inflammatory bowl disease, acute and chronic pain, rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g., asthma, adult respiratory distress syndrome, wound healing, and scar formation), as well as several other disoders and/or diseases (e.g., periodontal disease, dysmenorrhea, premature labor, brain edema following focal injury, diffuse axonal injury, and reperfusion injury).
In specific embodiments, the present invention encompasses methods and compositions (e.g., ACE-2 binding polypeptides that antagonize ACE-2 activity) for preventing, treating and/or ameliorating diseases or disorders associated with hypertension (e.g., accelerated hypertension, renal failure, vascular accidents, myocaridal infarction, and stroke).
In a specific embodiment, this invention also provides a method for preventing, ameliorating, or treating diseases and/or disorders associated with hypotension (e.g., shock, syncope, and intracranial hypotension) comprising, or alternatively consisting of, administering to an animal in which such inhibition or reduction is desired, an effective amount of angiotensin H, or an angiotensin II-like compound, and an effective amount of angiotensin 1-9, or an angiotensin 1-9-like compound.
In specific embodiments, the present invention encompasses methods and compositions (e.g., ACE-2 binding polypeptides that antagonize ACE-2 or ACE activity) for preventing, treating and/or ameliorating other diseases or disorders associated with vasoconstriction, comprising, or alternatively consisting of, administering to an animal in which such treatment, prevention, and/or amelioration is desired, an ACE-2 binding polypeptide in an amount effective to treat, prevent and/or ameliorate the disease or disorder.
The present invention further encompasses methods and compositions for inhibiting or reducing stenosis, including aortic stenosis, buttonhole stenosis, coronary ostial stenosis, double aortic stenosis, fish-mouth mitral stenosis, bronchial stenosis, hypertrophic pyloric stenosis, pyloric stenosis, infundibular stenosis, idiopathic hypertrophic subaortic stenosis, idiopathic subglottic stenosis, pulmonary stenosis, muscular subaortic stenosis, laryngeal stenosis, mitral stenosis, supravalvar and subvalvar stenosis, subvalvular and supravalvular stenosis, and tricuspid stenosis, comprising, or alternatively consisiting of, contacting an effective amount of ACE-2 binding polypeptide, wherein the effective amount of ACE-2 binding polypeptide inhibits or reduces ACE-2 mediated enzymatic action.
The present invention further encompasses methods and compositions for inhibiting or reducing pain, comprising, or alternatively consisting of, administering to an animal in which such inhibition or reduction is desired, an ACE-2 binding polypeptide in an amount effective to inhibit or reduce ACE-2 enzymatic activity.
The present invention further encompasses methods and compositions for inhibiting or reducing inflammatory reactions in various tissues comprising, or alternatively consisting of, contacting an effective amount of ACE-2 binding polypeptide, wherein the effective amount of ACE-2 binding polypeptide inhibits or reduces ACE-2 enzymatic activity.
In a specific embodiment, the present invention encompasses methods and compositions for inhibiting or reducing inflammatory reactions in smooth muscle tissues comprising, or alternatively consisting of, contacting an effective amount of ACE-2 binding polypeptide, wherein the effective amount of ACE-2 binding polypeptide inhibits or reduces ACE-2 enzymatic activity.
The present invention further encompasses methods and compositions for inhibiting or reducing abnormal histamine release comprising, or alternatively consisting of, administering to an animal in which such inhibition or reduction is desired, an ACE-2 binding polypeptide in an amount effective to inhibit or reduce ACE-2 enzymatic activity.
In another embodiment, the present invention encompasses methods and compositions for inhibiting or reducing vasoconstriction and/or other diseases or disorders associated with vasoconstriction comprising, or alternatively consisting of, administering to an animal in which such inhibition or reduction is desired, an ACE-2 binding polypeptide in an amount effective to inhibit or reduce ACE-2 enzymatic activity.
In yet another embodiment, this invention also provides a method for reducing or inhibiting diseases and/or disorders associated with aberrant action of ACE-2 comprising, or alternatively consisting of, administering to an animal in which such inhibition or reduction is desired, an ACE-2 binding polypeptide and an effective amount of an ACE inhibiting compound.
Additionally, this invention also provides a method for reducing or inhibiting diseases and/or disorders associated with aberrant action of ACE comprising, or alternatively consisting of, administering to an animal in which such inhibition or reduction is desired, an ACE-2 binding polypeptide and an effective amount of an ACE inhibiting compound.